Figure 1. Increased autophagic flux in thymocytes from lupus-prone mice compared with controls (A) A representative autophagosome is indicated by the white arrow (black scale bar: 500 nm). (B) Quantification by TEM of autophagic vacuoles for 50 thymocyte sections randomly selected on thymus sections from 8 week-old CBA/J (open circles) and 12 week-old BALB/c (filled circles) control mice (CTL) and lupus-prone mice MRLlpr/lpr (8 week-old) and NZB/W (12 week-old). Each point represents measurement for an individual mouse. Central bars refer to the mean and vertical bars stand for standard deviation. ns = non-significant using unpaired t-test. (C) LC3 conversion assessed by western immunoblotting. Dissociated thymocytes obtained from 8 week-old control CBA/J and lupus MRLlpr/lpr mice or from 12 week-old control BALB/c and lupus NZB/W mice were cultured at 37°C for 16h. When indicated, cells were treated (+) or not (-) during the last 4 h of the culture with 5 µg/mL pepstatin A and 5 µg/mL E64d to block lysosomal degradation. Cell lysates were resolved by SDS-PAGE, transferred onto PVDF membranes before staining with anti-LC3 Ab. Loading controls were performed by staining actin β-chain. Each immunoblot is representative of three experiments with identical results. LC3-II/β-actin band intensity ratios are indicated as numbers under each immunoblot. (D) LC3-II levels were evaluated by densitometry and normalized to β-actin band intensities for at least three other independent experiments (right panel). Autophagic flux measurement consists on a ratio between the values with and without protease inhibitors (= autophagic flux). Histogram bars represent the means of individual experiments with standard errors. *p < 0.05 and **p < 0.01 using unpaired t-test between control and lupus conditions.